title: Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.
abstract: Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.
authors.affiliation: Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, United States of America. jurat@utk.edu
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Jurat-Fuentes
authors.forename: Lohitash
authors.initials: L
authors.lastname: Karumbaiah
authors.forename: Siva Rama Krishna
authors.initials: SR
authors.lastname: Jakka
authors.forename: Changming
authors.initials: C
authors.lastname: Ning
authors.forename: Chenxi
authors.initials: C
authors.lastname: Liu
authors.forename: Kongming
authors.initials: K
authors.lastname: Wu
authors.forename: Jerreme
authors.initials: J
authors.lastname: Jackson
authors.forename: Fred
authors.initials: F
authors.lastname: Gould
authors.forename: Carlos
authors.initials: C
authors.lastname: Blanco
authors.forename: Maribel
authors.initials: M
authors.lastname: Portilla
authors.forename: Omaththage
authors.initials: O
authors.lastname: Perera
authors.forename: Michael
authors.initials: M
authors.lastname: Adang
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000633:toxicity*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D004731:Endotoxins / Q000633:toxicity*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006460:Hemolysin Proteins / Q000633:toxicity*; D007305:Insecticide Resistance / Q000187:drug effects*; D007814:Larva / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D007915:Lepidoptera / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000187:drug effects / Q000201:enzymology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase